. Human X-linked agammaglobulinemia (XLA) results from deficient function of Bruton's tyrosine kinase (Btk). In affected males, the production of B lineage cells is profoundly reduced resulting in life threatening humoral immunodeficiency. Definitive genetic therapy utilizing expression of wild type Btk in stem cells or B lineage progenitors would represent a significant advantage over the current supportive management of XLA. The Investigator proposes to establish pre-clinical cellular models for the genetic treatment of XLA through three aims. Aim 1. Development of a multistage human B cell culture system that can be used to evaluate reconstitution of B cell function in vitro. The Investigator will improve the derivation of B cells representing multiple stages of the B lineage from long-term human B progenitor cell cultures. This will be done by exploiting a range of culture modifications and signals that lead to activation, maturation, and production of secreted immunoglobulin by immature B cells. Soluble factors and cell surface ligands on stromal cells will be evaluated. The Investigator will also utilize in vivo transfer studies in SCID/NOD mice. Aim 2: Development and testing of the capacity of retroviral vectors capable of high level, sustained expression in hematopoietic stem cells, and of novel hybrid adenoviral/EBV episome vectors for Btk gene transfer and rescue of Btk signaling in vitro. Alternative gene transfer systems will be evaluated using a new signaling competent human B cell model developed by the Investigator's laboratory. This will allow the Investigator to rapidly test the transduction efficiency, and the levels and duration of protein expression of alternative vectors. In addition, the Investigator will directly evaluate the capacity of these vectors to rescue Btk dependent signaling events and determine the dosage of Btk required for functional reconstitution in human B cells. Aim 3. Evaluation of the capacity of those gene transfer vectors that successfully restore Btk signaling in vitro to reconstitute Btk dependent immune function in vivo in Btk-/- mice, and ultimately, in vitro in long term cultured XLA B cells. The Investigator will evaluate gene transfer using hematopoietic stem cells and growth factor expanded B progenitors from Btk-/-mice as target cell populations. Reconstitution of B lineage development and function will be evaluated using multiple measures of Btk dependent immune function. Those vectors demonstrating optimal restoration of Btk function in vivo will be utilized for gene transfer into stem cells or B progenitors in pre-clinical studies of patients with XLA.